When less is more - a fast TurboID knock-in approach for high-sensitivity endogenous interactome mapping

Area of Science:

• Cell Biology
• Molecular Biology
• Proteomics

Background:

• Proximity labeling is a key unbiased method for mapping protein interactomes.
• Generating cell lines for endogenous protein labeling is typically time-consuming and difficult.

Purpose of the Study:

• To develop and validate a rapid CRISPR-Cas9 knock-in pipeline for endogenous proximity labeling.
• To compare the efficacy of different labeling enzymes for endogenous protein tagging.
• To investigate the interactomes of adaptor protein (AP) complexes and clathrin.

Main Methods:

• Utilized a CRISPR-Cas9 knock-in strategy with antibiotic selection for rapid generation of tagged cell lines.
• Performed endogenous tagging of the µ subunit of the AP-1 complex with TurboID.
• Compared interactomes of different AP complexes and clathrin using proximity labeling.

Main Results:

• Endogenous TurboID tagging of AP-1 complex µ subunit identified known interactors and cargo proteins missed by overexpression.
• The knock-in strategy enabled comparative interactome analysis of AP complexes and clathrin.
• Generated specific lists of potential interactors and cargo proteins for distinct cellular sorting pathways.

Conclusions:

• The developed pipeline significantly simplifies endogenous proximity labeling experiments.
• This approach accelerates the process from CRISPR transfection to interactome data analysis to just over one month.
• Enables comprehensive mapping of protein interaction networks in native cellular environments.